The present invention relates to a tissue treatment composition, especially a tissue adhesive having improved properties and to the use of such compositions as anti-adherence or would healing compositions, as slow-release drug formulations, for coating tissues or prosthetic materials, and as carriers for cell transplants.
The use of blood coagulating substances for stopping bleedings and for sealing wounds has been known for a long time. Thus, the hemostatic effect of fibrin powder was reported about 80 years ago, and attempts were made to employ fibrin or fibrin patches to stop bleeding in brain and general surgery.
Today such use of fibrin as a biologic adhesive has been widely accepted and found application in many fields of surgery. Generally fibrin sealants are based upon the two components fibrinogen and thrombin. As these components mix a fibrin coagulum is formed in that the fibrinogen molecule is cleaved through the action of thrombin to form fibrin monomers which spontaneously will polymerize to form a three-dimensional network of fibrin, largely kept together by hydrogen bonding. This corresponds to the last phase of the natural blood clotting cascade, the coagulation rate being dependent on the concentration of thrombin used.
In order to improve the tensile strength, covalent crosslinking between the fibrin chains is provided for by including Factor XIII in the sealant composition. The strength of the fibrin clot is further improved by the addition of fibronectin to the composition, the fibronectin being crosslinked and bound to the fibrin network formed.
To prevent a too early degradation of the fibrin clot by fibrinolys, the fibrin sealant composition may comprise a plasminogen activator inhibitor or a plasmin inhibitor, such as aprotinin. Such an inhibitor will also reduce the fibrinolytic activity resulting from any residual plasminogen in the fibrinogen composition.
Similarly, compositions according to the invention which include hyaluronic acid (or other polysaccharides), may also comprise a hyaluronidase inhibitor such as one or more flavonoids (or corresponding inhibitors for other polysaccharides) in order to prevent premature degradation (i.e. to prolong the duration) of the hyaluronic acid component by hyaluronidase which is always present in the surrounding tissues. The hyaluronic acid may, as mentioned above, be crosslinked, a commercially available example being Hylan(copyright) (trademark, available from Biomatrix, Ritchfield, N.Y., USA). The hyaluronic acid compositions may e.g. have the form of gels, solutions, etc.
The results obtainable by fibrin sealants are basically:
(i) Hemostasis. The fibrin clot acts as a hemostatic barrier and reduces the risk of serum, lymph and liquor leakage. The hemostatic effect may be enhanced if the fibrin sealant is combined with a biocompatible solid flat material such as collagen.
(ii) Glueing. Due to its adhesive properties the fibrin sealant atraumatically connects tissues by forming a strong joint between them and adapts uneven wound surfaces. This glueing effect is increased by fibronectin being bound to exposed collagen.
(iii) Wound healing. The fibrin sealant promotes the ingrowth of fibroblasts which in combination with efficient hemostasis and adhesion between the wound surfaces provides for an improved healing process. Wound healing promoted by fibrin sealants results in strong scar formation and does not prevent the formation of adhesions. The use of the compositions according to the invention as an anti-adherence/wound healing composition does, however, result in a normal (regenerative) tissue rather than scar tissue, i.e. optimal wound heaing. Furthermore, such compositions also reduce the inflammatory response as appears from the test results reported in Table 4 below.
Fields of application include among others: ear, nose and throat surgery, general surgery, dentistry, neurosurgery, plastic surgery, thorax and vascular surgery, abdominal surgery, orthopaedics, accident surgery, gynaecology, urology, and opthalmology. Fibrin sealants have also been used for local application of drugs, such as antibiotics, growth factors and cytostatics.
Commercial fibrin glues (prepared from human plasma) are available under the trade names Tissucol, Tisseel and Fibrin-Kleber Humano Immuno (Immuno AG, Vienna, Austria) as well as Beriplast (Behringwerke AG, Marburg, Germany) (these trade names being registered trademarks in several countries). Tisseel(trademark) is a two-component kit containing a fluid thrombin component including calcium chloride and a somewhat more viscous fibrinogen component including factor XIII, fibronectin, aprotinin and plasminogen. The two components are delivered deep frozen in two separate syringes, or as two lyphilized powders with corresponding aprotinin and calcium solutions as solvents. As explained above the fibrin sealant consolidates when the two components are combined due to fibrin monomer aggregation. The setting rate is dependent on the thrombin concentration and varies from a few seconds (high thrombin concentration) to a couple of minutes (low thrombin concentration).
However, an important and well known disadvantage of the known preparations resides in the water-like fluidity of the components when applied, which leads to considerable handling difficulties of the glue. Efforts have been made to overcome this problem and facilitate the mixing of the components by the development of particular application modes such as a double-syringe applicator (e.g. that supplied under the trade name Duploject(copyright), Immuno AG, Vienna, Austria, and which is disclosed in e.g. U.S. Pat. No. 4,359,049, or a special spray system as disclosed in e.g. EP-A-156 098). The basic problem with a low viscosity glue still remains, however. Firstly, a non-viscous or low viscosity glue is unsuitable for use on non-horizontal surfaces since it will run off before setting. Secondly, there is a definite risk of a non-viscous or low vicosity glue running off to sites where it is unwanted and where it might cause complications. This is particularly the case in vascular surgery since the fluid glue may reach inside the vessels before it sets and thereby cause thromboembolic complications. An instantantaneously setting fibrin glue (containing a high concentration of thrombin), on the other hand, cannot be used where the parts to be sealed require subsequent adaptation.
A different approach has been disclosed by i.a. Bass et al in J. Vasc. Surg. May 11, 1990 (5):718-25, which is incorporated herein by reference. This paper discloses a technique called laser tissue soldering (or welding), wherein a laser energy absorbing dye (chromophore) and fibrinogen are soldered by means of a laser to produce a strong anastomosis which is said to be i.a. faster healing than a conventional sutured anastomosis. Similar coagulation and/or bonding effects can be achieved with other proteins and energy sources.
It is an object of the present invention to provide an improved fibrin glue which is devoid of the above low viscosity problem, and which promotes wound healing without scar formation or development of adhesions. This object is achieved by including in a fibrin glue composition of the above mentioned type a viscosity increasing amount of a biodegradable and biocompatible polymer capable of forming a viscsous aqueous solution. In accordance with the present invention it has thus been found that by the addition of such a viscosity enhancing polymer, the glue composition will obtain a viscosity adequat to facilitate and improve the handling and application thereof, while not negatively affecting the favourable properties of the fibrin glue. For wound healing and anti-adherence purposes the adhesive properties may, however, be less pronounced, or even missing.
Accordingly, the present invention relates to a tissue treatment composition comprising (i) fibrin or fibrinogen and (ii) a biodegradable and biocompatible polymer capable of forming a viscous aqueous solution, optionally also other proteins.
One use form of the present tissue adhesive composition is thus an improved fibrin sealant or glue which upon use exhibits viscosity characteristics permitting easy and safe application thereof at a desired location or site.
In another use form the present tissue treatment composition comprises a therapeutical substance and constitutes a pharmaceutical composition for local administration of the therapeutical substance. In a particular embodiment the therapeutical substance is the viscous polymer itself or a species coupled thereto as will be described in more detail below.
Still another use form of the present tissue treatment composition is a wound healing and an anti-adherence composition, the high molecular composition conferring such adherence-preventing properties to the composition that it may be used for preventing the adherence of adjacent tissues in surgical procedures. Related to such anti-adherence use is the use of the present tissue treatment composition for wound healing. By, for example, glueing wound edges with the tissue treatment, neat scars will be obtained. Further, cellular translplants, in particular dermal transplants, will heal faster. This would, of course, be of particular interest in plastic surgery.
The above mentioned biodegradable and biocompatible polymer capable of forming an aqueous solution may be selected from a wide variety of substances (including substances which will be available in the future) and the selection thereof can readily be made by the person skilled in the art.
A preferred group of said biodegradable and biocompatible polymers, hereinafter frequently referred to as viscosity enhancing polymers, consists of high molecular polyglycans or polysaccharides. Exemplary of such polysaccharides for the purposes of the invention are xanthan, dextran, cellulose and proteoglycans, especially hyaluronic acid, and salts and derivatives thereof. As examples of cellulose derivatives may be mentioned methyl cellulose, carboxymethyl cellulose (CMC) and hydroxy-propylmethyl cellulose (HPMC), just to mention a few thereof. Examples of viscosity enhancing polymers other than high molecular polysaccharides are gelatin and polyvinylpyrrolidone.
A preferred polysaccharide/polyglycan is hyaluronic acid and salts and derivatives thereof. Sodium hyaluronate is a high molecular weight linear polysaccharide built up of repeating disaccharide units. It exists in the extracellular space of all tissues and has the same simple chemical structure n all species. Hence, the application of a purified preparation of hyaluronate results in but a temporary increase of the local concentration of endogenous material and its utilization in the composition will therefore not have any detrimental physiological effects. In solution the hyaluronate adopts a conformation of very extended random coils, that already at low concentrations entangle into a flexible molecular network that gives hualuronate solutions interesting rheological properties that are useful for the present purposes.
The visco-elastic properties of sodium hyaluronate has lead to its clinical use as spacer and to facilitate operative procedures in the field of eye surgery. It has also been demonstrated to be biologically active in enhancing epithelial regeneration of the ear tympanic membrane and to inhibit the ingrowth of vascular endothelial cells. Further, it plays a role in wound healing, influencing the migration of granulation tissue cells and reduces the amount of adhesions formed after surgery. The bioavailability of sodium hyaluronate per se is, however, limited due to its rapid turnover and short half-life.
When the tissue treatment composition is used as an improved tissue adhesive, the proportion of the viscosity enhancing polymer in the fluid fibrin glue as applied should be selected to provide an appropriate viscosity for the intended application while not adversely interfering with the fibrin clotting, and will depend on the polymer and the particular tissue adhesive composition to be produced. Suitable initial viscosities of the final solution mixture of the total composition for each particular application may readily be established by the skilled person, but will generally be in the range of about 500 to about 1,000,000 centipoises (cP), preferably about 1,000 to about 500,000 centipoises. The term xe2x80x9cfinal solution mixturexe2x80x9d as used herein does not necessary mean a homogeneous state. On the contrary, depending on the mixing procedure, the mixture will in many cases not reach a homogeneous or uniform state before clotting. As is well known to the person skilled in the art, the viscosity is correlated to concentration and limiting viscosity number, xcex70=(Conc.xc3x97[xcex7])3,6/10. Modified after Morris et al, Carbohydrate polymers, Vol. 1, 1981, p. 5-21. From [xcex7] we get the molecular weight using Cleland""s formula for [xcex7]=kxc3x97average molecular weightk1, Cleland et al, Biopolymers, Vol. 9, 1970, p. 799-80.
Like the prior art fibrin sealants the tissue adhesive composition of the present invention may comprise additional constituents. Thus, in addition to sealer protein and viscosity enhancing polymer, such as e.g. high molecular polysaccharide, the Composition will preferably comprise Factor XIII and/or fibronectin and/or plasminogen. Advantageously, the composition will also include clotting enzyme, i.e. thrombin, especially in combination with bivalent calcium, such as calcium chloride. The concentration of calcium chloride will then vary) e.g. between 40 mM to 0.2 M depending on the specific purpose of the tissue adhesive composition, high concentrations of calcium chloride inhibiting fibroblast growth and therefore being preferred for anti-adherence applications (along with absence of fibronectin which stimulates the ingrowth of fibroblasts). It may further be valuable to include a fibrinolysis inhibitor, such as a plasmin inhibitor, e.g. aprotinin, aprilotinin, alpha-2-antiplasmin, alpha-2-macroglobulin, alpha-1-antitrypsin, epsilon-aminocaproic acid or tranexamic acid, or a plasmin activator inhibitor, e.g. PAI-1 or PAI-2.
While the proportions of the previously known ingredients in the tissue adhesive compositions of the invention may be selected with guidance of prior art compositions, the necessary amount of the viscosity enhancing polymer can readily be determined by a person skilled in the art depending on the particular polymer and the intended use form. Thus, if the concentration and/or molecular weight of the viscosity enhancing polymer is too low, the viscosity increase will be insufficient, and a too high concentration and/or molecular weight will inhibit the fibrin polymerization and the adhesion to the tissue.
By increasing the thrombin concentration, the polymerization of fibrinogen may be speeded up with a consequential influence on the time until the glue sets. At low thrombin concentrations the fibrin glue composition will remain more or less fluid for several minutes after application. A further beneficial effect of increasing the viscosity with a viscosity enhancing polymer in accordance with the invention is therefore the possibility to use lower concentrations of thrombin, which is required in situations where the parts to be sealed require subsequent adaptation even on non-horizontal surfaces.
The tissue treatment composition of the present invention may be presented in the same type of preparations as the prior art fibrin sealants. In an advantageous embodiment the tissue adhesive is therefore a two-component preparation, one component comprising the blood clot protein(s) and the other comprising thrombin and bivalent calcium as well as possible additives including fibrinolysis inhibitors. The viscosity enhancing polymer may be contained in one or both of the two components depending on the intended use of the tissue adhesive. While in the case of a fibrin glue the viscosity enhancing polymer may be contained in either or both of the two components, it is for other applications preferably associated with the fibrin or fibrinogen component. It is, of course, at least theoretically, also possible to provide the viscosity enhancing polymer as a separate component. The components may be provided in deep frozen solution form or as lyophilized powders, to be diluted prior to use with appropriate aqueous solutions, e.g. containing aprotinin and calcium ions, respectively.
The tissue treatment composition of the invention may also be used in various combinations as is per se known in the art. For example, with reference to the above mentioned two-component embodiment, one component may be provided in a biocompatible solid matrix material as a prefabricated unit and the other (activating) component may be added at the time of use. The viscosity enhancing polymer may then be provided together with any one or both of said components.
In such an embodiment the tissue adhesive of the invention may include a tissue-compatible flat matrix material, such as a non-woven fabric, into which the blood coagulation substance, the viscosity enhancing polymer, e.g. high molecular polysaccharide, and optional additional constituents are impregnated. In a variation the viscosity enhancing polymer is added together with the thrombin. In another variation the matrix material is impregnated with the thrombin, and the blood coagulation substance is added together with the viscosity enhancing polymer at the time of use. Such a non-woven fabric may, for example, be a glycoprotein, such as collagen (preferably porous), globulin, myoglobulin, casein or albumin; gelatin; silk fibroin or a polysaccharide, such as cellulose; or mixtures thereof. Such an embodiment will, for instance, be particularly useful for stopping bleedings and covering wounds. It is to be noted, however, that, as will be readily understood, for anti-adherence purposes a material like collagen having adhesion enhancing properties would not be appropriate; cellulose e.g. being a more suitable material in this respect. Such an impregnated flat material is advantageously provided in lyophilized form.
In another embodiment the tissue treatment composition is provided as a film or sheet for surgical use comprising a non-crosslinked combination of fibrin and viscosity enhancing polymer.
The tissue treatment composition of the present invention may, of course, be used in all other preparations in which the prior art fibrin glues have been presented, e.g. as an implantation material for joint cartilage and bone defect repair material in combination with embryonic chondrocytes or mesenchymal cells, such as described for a conventional fibrin glue in e.g. U.S. Pat. No. 4,642,120.
As already mentioned above the present tissue treatment composition, e.g. in any one of the above described embodiments, may be used for the application of a pharmaceutically active substance. By incorporating a drug, such as an antibiotic, a growth factor, etc. into the tissue adhesive it will be enclosed in the fibrin network formed upon application of the tissue adhesive. It will thereby be ensured that the drug is kept at the site of application while being controllably released from the composition, e.g. when used as ocular drops, a wound healing preparation, etc.
As also mentioned above the pharmaceutically active substance to be released from the present tissue adhesive composition may be the viscosity enhancing polymer in itself or a substance coupled thereto.
A specific example of such a viscosity enhancing polymer fulfilling the viscosity enhancing requirement as well as having therapeutical and pharmaceutical utility, and for which it may be desired to sustain the bioavailability, is hyaluronic acid and salts and derivatives thereof which are easily soluble in water and, as mentioned previously, have an extremely short biological half-life. The tissue treatment composition of this invention thus constitutes an advantageous slow-release preparation for proteoglycans such as hyaluronic acid and its salts and derivatives, and considerably increases the bioavailability thereof.
The tissue treatment composition of the present invention may, for example, be prepared and provided in administration forms in analogous manner as the prior art tissue adhesives.
It should be emphasized that the compositions are not restricted to the adhesive properties, but non-adhesive compositions are also included, especially when the compositions primarily are intended for wound healing. The latter compositions may in particular include non-adhesive proteins such as albumin and/or growth factors. Substantially non-adhesive compositions may also be obtained when the polymer part of the composition inhibits the adhesive properties of the protein part. It should in this context be emphasized that the invention comprises both adhesive and substantially non-adhesive compositions, although it has for simplicity reasons often has been referred to as an xe2x80x9cadhesivexe2x80x9d in this specification, including the Examples.
In the following the invention will be described in more detail by way of non-limiting examples. In one example the gluing properties of an embodiment of the tissue adhesive composition are tested in animal experiments, reference being made to the accompanying drawing in which the only figure is a schematic side view of a clamped vessel with three sutures. A second example describes the preparation of another embodiment of tissue adhesive composition. A third example illustrates the use of the tissue treatment composition as a controlled release preparation, and a fourth example shows the properties of the tissue treatment composition as an anti-adhesion and wound healing promoting agent.